List of Titles

Evaluating baculovirus mixtures against false codling moth Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae)

Authors: Tole, Siviwe
Date: 2024-10-11
Subjects: False codling moth Biological control , Baculoviruses , Integrated pest management , Natural pesticides , Granulovirus
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/463996 , vital:76464
Description: False codling moth (FCM), Thaumatotibia leucotreta, is an important pest of citrus, stone fruit, avocados, peppers, and other important agricultural crops in southern Africa. Baculovirus-based biopesticides are components in an integrated pest management (IPM) programme to manage the pest in the field. Cryptogran™ and Cryptex™ which are CrleGV-SA based-biopesticides have been effective in the control of T. leucotreta for the past 15 years. Recently, CrpeNPV-based Multimax™ and Codlmax™ have been commercialised to control T. leucotreta and other important agricultural pests. Despite these viruses being relatively host-specific and safe to humans and animals in comparison to chemical insecticides, their application is hindered by their slow speed of kill, sensitivity to UV light, and the potential for insect resistance. Research investigating the effects of mixed baculoviral interactions against target pests has been a growing field of interest due to their potential to overcome such shortcomings. Previous studies using a combination of CrleGV-SA and CrpeNPV against T. leucotreta observed a reduction in lethal concentration in laboratory bioassays, indicating that such mixtures may have the potential for application in the field. This has led to the motivation to investigate further interactions between CrleGV-SA in combination with CrpeNPV, CpGV-M, and HearNPV-Au to understand better how these viruses interact and to determine whether synergistic, additive, or antagonistic interactions can occur against T. leucotreta. The outcome of these interactions will inform researchers and farmers about best practices concerning these viruses should they be combined against T. leucotreta in the future. Prior to performing mixed baculovirus infections in laboratory bioassays, oligonucleotides targeting unique regions in the viral genomes of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au were designed using Primer-BLAST. The specificity of these oligonucleotides was further tested in silico using Geneious R11 software (11.1.5). The stocks of CrpeNPV, CpGV-M, and HearNPV-Au were purified using crude OB extraction from diseased C. peltastica, C. pomonella, and H. armigera larval cadavers provided by River Bioscience (Pty) Ltd (Gqeberha, South Africa). The stock of CrleGV-SA was purified using crude OB extraction from infected T. leucotreta cadavers. Subsequently, the unique oligonucleotides were used in PCR assays to detect if the samples contained the baculoviruses of interest. Amplicons of the expected sizes were generated indicating the presence of CrleGV-SA, CrpeNPV, CpGV-M, and HearNPV-Au in each of the samples. The OBs were counted using darkfield microscopy and a counting chamber before the single and mixed infections were initiated against T. leucotreta neonate larvae. Surface-dose biological assays were used to evaluate the relative virulence in terms of lethal concentration of CrleGV-SA, CrpeNPV, and CpGV-M, alone against T. leucotreta. After 7 days, the dose mortality data was analysed using “drc” in R studio and the LC50 and LC90 were compared amongst each virus. The CrleGV-SA treatment was estimated to be the most virulent in comparison to CrpeNPV and CpGV-M. A dose discriminate assay confirmed that HearNPV does not cause mortality in T. leucotreta. Similarly, the relative virulence in terms of lethal concentration of CrleGV-SA in various ratios in combination with CrpeNPV, CpGV-M, and HearNPV-Au was determined using 7-day surface dose biological assays. The CrleGV/CrpeNPV was the most virulent mixture with lower LC50 and LC90 values measured in comparison to CrleGV/CpGV and CrleGV/HearNPV, respectively. The Tammes Bakuniak graphic method confirmed the CrleGV/CrpeNPV, CrleGV/CpGV, and CrleGV/HearNPV mixtures to be antagonistic against T. leucotreta neonate larvae in terms of lethal concentration. The last aspect of the study was to determine the probable cause of larval death. A modified CTAB protocol was used to extract genomic DNA from neonate-sized T. leucotreta cadavers collected in single and mixed infection assays. The gDNA served as templates in PCR assays using the unique oligonucleotides. In single infections, the presence of CrleGV-SA in CrpeNPV and HearNPV inoculated larvae was observed. The results suggest possible covert infections of CrleGV-SA in the T. leucotreta colony which may be caused by virus infection or an unknown stress factor. The results from the mixed infections showed the presence of each virus in all replicates except for the CrleGV/CpGV and CrleGV/HearNPV mixtures. In the CrleGV/CpGV mixture, only CrleGV-SA was present in the last replicate, suggesting a possible competition for host resources. In the CrleGV/HearNPV mixture, only CrleGV-SA was detected in all 3 replicates, suggesting that HearNPV did not have any effect and the larvae died of the CrleGV-SA infection. This is the first study to report mixtures of CrleGV-SA in combination with CpGV-M and HearNPV-Au against T. leucotreta neonate larvae. Despite the antagonistic interactions observed in the evaluated mixtures, this study has laid a foundation to further investigate how these viruses interact in dual infections for the improved control of T. leucotreta. This may be done by evaluating different ratios and combinations of baculoviruses to those used in this study. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
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Investigation into methods of recovering campylobacter spp. from river water samples

Authors: Ngoni, Nandipha
Date: 2023-10-13
Subjects: Campylobacter jejuni , Stream chemistry , Organic water pollutants South Africa Eastern Cape , Water quality Measurement , Turbidity , Physicochemical process
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/424177 , vital:72130
Description: Campylobacter species are slender, gram-negative, rod-shaped, spiral- or curved-shaped with single or pairs of flagella. They are the leading cause of diarrheal disease globally, consumption of and contact with water contaminated by faeces is a major risk factor for transmission of these organisms to humans. Rivers used for recreation and domestic and agricultural activities represent all the risk factors for Campylobacter spp. pollution and human exposure. Campylobacter spp. However, effective methods to recover Campylobacter spp. from river water samples are lacking, indicating the need for the development of more efficient methods of detection and isolation of these organisms from environmental water samples. Campylobacter detection in a water sample is critical to ascertain potential risks to humans. The aim of this study was to determine a suitable method for the detection of Campylobacter spp. from river water samples and the objectives were to (i) to evaluate the performance of different methods used for the recovery of Campylobacter spp. from environmental water samples based on Campylobacter colony count and PCR identification results, (ii) isolate and enumerate Campylobacter cells from river water samples, and (iii) identify Campylobacter spp. in river water samples. The Bloukrans River was chosen for this study because it is suspected to be contaminated by faecal inputs from nearby informal settlements without adequate sanitation, as well as untreated/insufficiently treated effluents from nearby wastewater treatment plants. First, the physicochemical quality of the river water and the presence of faecal contamination were assessed to confirm suitability for Campylobacter spp. survival and presence. Then different approaches to sample, concentrate and recover Campylobacter spp. from river water samples were assessed. The different methods assessed were (i) direct enrichment of water samples without prior concentration, (ii) prior concentration of water samples by centrifugation followed by membrane filtration of supernatant, and after that, pooling the residue and pellet together for enrichment, (iii) sampling by the Moore Swab technique. For all three methods, enrichment in Bolton broth supplemented with Bolton antibiotics was conducted. This was followed by plating on modified cefoperazone charcoal deoxycholate agar (mCCDA) and incubation under a microaerophilic atmosphere at 42°C for 48 h. Colony morphology, Gram staining and polymerase chain reaction (PCR) were used to identify and characterize the microorganisms. The growth of blue colonies on the mFc agar surface confirmed presence and faecal pollution of the Bloukrans River. The physicochemical properties, based on the range of pH measured at different sites of the river (between acidic 3.45 to 6.42 and alkaline 7.2 to 8.74) indicate that Campylobacter spp. can thrive in the river. Based on the results from enumeration and sequencing of colonies recovered by each method, it was discovered that the most suitable method to recover Campylobacter spp. from river water samples is by prior centrifugation (14,000 × g for 30 minutes) followed by membrane filtration of the supernatant, and subsequent pooling of the residue and pellet. The pooled residue and pellet might have increased Campylobacter spp. concentrations aiding more growth during the enrichment of Campylobacter spp. from the river water samples. Results from enumerating Campylobacter spp. cells from river water samples indicate that Campylobacter spp. are present in Bloukrans River. The sequence obtained from the PCR product indicates that the species found were Campylobacter jejuni (96% homology as evaluated by BLAST). This study provided a procedure effective for obtaining a satisfactory quantitative recovery of Campylobacter spp. from environmental waters, a critical need for quantitative microbial risk assessment studies. , Thesis (MSc) -- Faculty of Science, Institute for Water Research, 2023
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The development and op timisation of a Theiler’s murine encephalomyelitis virus antiviral assay

Authors: Naidoo, Urisha Tirah
Date: 2023-10-13
Subjects: Theiler's encephalomyelitis virus , Picornaviruses , Antiviral agents , Immunofluorescence , Western immunoblotting , Drug development
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/424677 , vital:72174
Description: Picornaviruses belong to the Picornaviridae family which are one of the largest and most diverse family of RNA viruses that cause a broad spectrum of infections in both humans and animals. These diseases range from severe infections such as poliomyelitis, meningitis, myocarditis to mild illnesses such as the common cold. Picornavirus outbreaks are a worldwide threat as they are continuously occurring. A recent outbreak of foot-and-mouth disease caused by a picornavirus occurred in South Africa, resulting in a temporary ban on the movement of cattle. Currently, the FDA has not approved any antiviral drugs against this virus, increasing the urgency for identifying effective antivirals. Picornaviruses have similar genomes and capsid organisation as such, those that are non-hazardous to humans can be used as a model system. A Theiler’s murine encephalomyelitis virus (TMEV) strain GDVII and Baby Hamster Kidney fibroblasts (BHK-21 cells) was used as a replication system to develop and optimise a medium-throughput antiviral screening assay. The TMEV GDVII replication system in BHK-21 cells was validated, and preliminary experiments were performed that were necessary for the development of the TMEV GDVII antiviral assay. This was achieved by conducting a CPE assay to visually monitor the onset and development of CPE induced by TMEV GDVII. Plaque assays accurately quantified the number of infectious virus particles required for calculating the MOI in downstream experiments. Lastly, indirect immunofluorescence and Western blot analysis detected the expression of viral proteins using previously generated antibodies against the TMEV GDVII VP1 capsid and 2C protein, thereby confirming infection in BHK-21 cells. The development of robust and reproducible assays is an essential component in antiviral drug discovery. Therefore, the confirmed replication system was then used as a foundation to develop a medium-throughput CPE-based TMEV GDVII antiviral assay whereby the parameters were optimised to produce one of high quality. Firstly, the quantitation of viral-induced CPE was examined and confirmed in a 96-well plate using resazurin as a cell viability indicator. Each parameter was tested at varying conditions, and the optimal was concluded as 2 % FBS in the assay media, a 15 000 cells/well seeding density, infecting the cells with TMEV GDVII at an MOI of 0.00625 and measuring resazurin at an endpoint of 72 hpi. Furthermore, the parameters were ii validated by calculating the Z’- factor, which consistently produced scores above 0.5, indicative of a reliable, robust, reproducible antiviral assay. Currently, there are no inhibitors against TMEV GDVII that have been reported or confirmed in cell lines, animal models or clinical trials. Therefore, once the optimal assay parameters were selected, it presented an opportunity to assess whether potential compounds, including itraconazole (ITZ) and dipyridamole (DIP), possessed antiviral activity that could firstly, be utilised as a control inhibitor when screening compounds against TMEV GDVII and secondly, contribute to research on this virus. Additionally, the previously produced anti-TMEV GDVII capsid antibody was shown to neutralise viral infection and was also included as a potential control. The sensitivity of the cells towards DMSO, a solution in which the compounds were solubilised, was first investigated. It was found that concentrations above 1 % are toxic to the cells; as such, the final DMSO concentrations were always kept below 1 % when screening compounds. Lastly, the generation of dose-response curves aided in the conclusion that the antibody was the most suitable control inhibitor as it displayed potent antiviral activity and no cytotoxicity towards the cells. In contrast, ITZ and DIP did not possess effective antiviral action and were toxic to cells at high concentrations. Finally, after all the components of the medium-throughput TMEV GDVII antiviral assay were identified, it was possible to screen 24 compounds from a coumarin and marine natural product library for cell cytotoxicity and antiviral activity. After generating dose-response curves, it was concluded that no compound effectively inhibited virus-induced CPE, and most were toxic to cells at relatively high concentrations. In conclusion, this is the first study that describes the development and optimisation of a robust medium-throughput CPE-based antiviral assay that has immense potential to screen other libraries of compounds for antiviral activity against TMEV GDVII. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2023
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Development and optimisation of a qPCR assay for the enumeration of Cryptophlebia leucotreta granulovirus (CrleGV) used for commercial applications

Authors: Mela, Thuthula
Date: 2022-10-14
Subjects: Cryptophlebia leucotreta granulovirus , Cryptophlebia leucotreta , Late expression factor 8 (LEF-8) , Late expression factor 9 , Dark field microscopy , Genomic DNA , Polymerase chain reaction , Plasmids
Language: English
Type: Academic theses , Master's theses , text
Identifier: http://hdl.handle.net/10962/362949 , vital:65377
Description: The citrus industry contributes significantly to the South African agricultural sector. Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is highly important to the South African citrus industry as it is classified as a phytosanitary pest by most international markets. Thaumatotibia leucotreta has caused an estimated annual loss of up to R100 million to the industry. In order to control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been used. One of the components of this programme is Cryptophlebia leucotreta granulovirus (CrleGV), which has been formulated to a registered biopesticide namely Cryptogran and has been successfully applied in the field for over 15 years. To use CrleGV as biopesticides, quantification of the viral particles is required to perform bioassays for field trials and formulation, among other applications. Darkfield microscopy is a traditional method used for the quantification of CrleGV; however, the method is characterised as being subjective, tedious, labour intensive, and time-consuming. This study aims to develop and optimise a qPCR technique to accurately quantify CrleGV-SA OBs using plasmid DNA for downstream applications. Firstly, lef-8, lef-9, and granulin conserved genes from CrleGV-SA and CrleGV-CV3 genome sequences were analysed by performing multiple alignments to evaluate the degree of identity between these genes. This was done to design two sets of oligonucleotides (internal and external) from regions with the highest identity. Subsequently, in silico testing was done to evaluate the designed oligonucleotides to determine whether they specifically bind to the selected target regions. Secondly, three sets of DNA plasmids (pJET1.2-Gran, pJET1.2-lef-9, and pJET1.2-lef-8) were constructed, each containing a target region for either granulin, lef-9, and lef-8 genes for use as standards in a downstream qPCR assay. This was achieved by first extracting gDNA from CrleGV-SA OBs and using the gDNA as a template to PCR amplify the target regions of the selected gene regions with the designed oligonucleotides. Subsequently, the PCR amplified regions were then directly ligated into the pJET1.2/blunt vector, and the plasmids were confirmed by colony PCR, restriction enzyme digestion, and Sanger sequencing. Thirdly, two different methods of CrleGV-SA gDNA extraction were compared to determine which method has the best yields in terms of concentration. The extraction methods compared were the Quick-DNA Miniprep Plus kit according to manufacturer’s instructions (Method 1a), pre-treatment with Na2CO3 prior to using the Quick-DNA Miniprep Plus kit (Method 1b), pre- treatment with Na2CO3, and neutralisation with Tris-HCl prior to gDNA extraction using the Quick-DNA Miniprep Plus kit (Method 1c) and the CTAB method (Method 2). The gDNA concentration and purity for all samples were determined using a Nanodrop spectrophotometer. Method 1c (Na2CO3 and Tris-HCl pre-treated plus Quick-DNA Miniprep Plus kit) was the most efficient at extracting genomic DNA compared with the other methods, resulting in the highest DNA concentration in short processing time. Fourthly, plasmid standards were evaluated for use in the qPCR assay. This was done as it was important to consider the efficacy of the oligonucleotides; including the ability of the oligonucleotides to anneal to the appropriate segment of DNA without extensive formation of oligonucleotides dimers, non-specific annealing, or formation of secondary structure. In addition, it was done to ensure that highly accurate standard curves were generated. The standard curves were to be utilised in the downstream qPCR assay to determine the quantity of test samples by interpolation, reading from the values within the standard curve. Lastly, darkfield microscopy and qPCR methods of enumeration were compared to verify their accuracy and determine the most consistent and comparable method. This was achieved by quantifying the purified, crude-purified, and viral formulated CrleGV-SA suspensions using these methods. Subsequently, a statistical analysis was conducted to compare the results produced by the two enumeration methods. The obtained results showed that the granulin, lef- 8 and lef-9 qPCR values did not significantly differ from the darkfield microscopy results. The findings of this study revealed that the two assays, lef-8 qPCR and lef-9 qPCR, were more robust, sensitive, and efficient for the quantification of CrleGV-SA. Thus, this study has successfully developed a qPCR assay that is comparable with the traditional darkfield microscopy counting technique. This is the first study to use the qPCR technique to enumerate CrleGV-SA using plasmid standards. The developed qPCR assay is reliable, rapid, and cost- effective and has a great potential to be used as an alternative method to darkfield microscopy in the laboratory and commercial settings. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2022
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An investigation into yeast-baculovirus synergism for the improved control of Thaumatotibia leucotreta, an economically important pest of citrus

Authors: Van der Merwe, Marcél
Date: 2021-10-29
Subjects: Baculoviruses , Cryptophlebia leucotreta , Yeast , Natural pesticides , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control , Thaumatotibia leucotreta‎
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/191236 , vital:45073
Description: A mutualistic association between Cydia pomonella and yeasts belonging to the genus Metschnikowia has previously been demonstrated. Larval feeding galleries inoculated with M. andauensis, reduced larval mortality and enhanced larval development. Additionally, adult C. pomonella female oviposition preference was also shown to be influenced by the volatiles produced by M. andauensis. This mutualistic relationship was manipulated for biological control purposes, by combining M. pulcherrima with the baculovirus Cydia pomonella granulovirus. The combination of M. pulcherrima with brown cane sugar and CpGV in laboratory assays and field trials resulted in a significant increase in larval mortality. A similar observation was made when M. pulcherrima was substituted for Saccharomyces cerevisiae. This indicates that yeasts harbour the potential for use in biological control, especially when combined with other well-established biocontrol methods. Thaumatotibia leucotreta is a phytophagous insect endemic to southern Africa. It is highly significant to the South African citrus industry due to its classification as a phytosanitary pest by most international markets. An integrated pest management programme has been implemented to control T. leucotreta. The baculovirus Cryptophlebia leucotreta granulovirus forms one component of this programme and is highly effective. In this study, we proposed to determine which yeast species occur naturally in the gut of T. leucotreta larvae and to examine whether any of the isolated yeast species, when combined with the CrleGV-SA, enhance its effectiveness. Firstly, Navel oranges infested with T. leucotreta larvae were collected from geographically distinct citrus-producing regions across South Africa. This led to the isolation and identification of six yeast species from the gut of T. leucotreta larvae via PCR amplification and sequencing of the internal transcribed spacer region and D1/D2 domain of the large subunit. Six yeast species were identified, viz. Meyerozyma guilliermondii, Hanseniaspora uvarum, Clavispora lusitaniae, Kluyveromyces marxianus, Pichia kudriavzevii and Pichia kluyveri. Additionally, Saccharomyces cerevisiae was included as a control in all trials due to its commercial availability and use in the artificial diet used to rear T. leucotreta. Secondly, larval development and attraction assays were conducted with the isolated yeast species. Thaumatotibia leucotreta larvae that fed on Navel oranges inoculated with M. guilliermondii, P. kluyveri, H. uvarum, and S. cerevisiae had accelerated developmental periods and reduced mortality rates. Additionally, it was demonstrated that T. leucotreta neonates were attracted to YPD broth cultures inoculated with P. kluyveri, H. uvarum, P. kudriavzevii and K. marxianus for feeding. Thirdly, oviposition preference assays were conducted with adult T. leucotreta females to determine whether the isolated yeast species influence their egg-laying in two-choice and multiple-choice tests. Navel oranges were inoculated with a specific yeast isolate, and mated adult females were left to oviposit. Meyerozyma guilliermondii, P. kudriavzevii and H. uvarum were shown to influence adult T. leucotreta female oviposition preference in two-choice tests. However, multiple-choice tests using the aforementioned yeast species did not mimic these results. Lastly, a series of detached fruit bioassays were performed to determine the optimal yeast:virus ratio, test all isolated yeast species in combination with CrleGV-SA and to further enhance yeast/virus formulation through the addition of an adjuvant and surfactant. CrleGV-SA was applied at a lethal concentration that would kill 50 % of T. leucotreta larvae. The optimal yeast concentration to use alongside CrleGV-SA was determined. Pichia kluyveri, P. kudriavzevii, K. marxianus and S. cerevisiae in combination with CrleGV-SA increased larval mortality compared to CrleGV-SA alone. The inclusion of molasses and BREAK-THRU® S 240 to P. kudriavzevii and S. cerevisiae plus CrleGV-SA formulations greatly enhanced their efficacy. Additionally, semi-field trials were initiated using P. kudriavzevii and S. cerevisiae, with promising preliminary results being obtained, although more replicates need to be performed. The experiments performed in this study provide a platform for further research into the application of a yeast/virus combination as a novel control and monitoring option for T. leucotreta in the field. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2021
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Baculovirus synergism for improved management of false codling moth Thaumatotibia leucotreta Meyr. (Lepidoptera: Tortricidae)

Authors: Taylor, David Graham
Date: 2021-04
Subjects: Baculoviruses , Cryptophlebia leucotreta , Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Codling moth , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
Language: English
Type: thesis , text , Masters , MSc
Identifier: http://hdl.handle.net/10962/176942 , vital:42774
Description: Baculoviruses are an environmentally friendly and effective agent for managing lepidopteran pests. This includes the management of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), a serious pest of citrus in Southern Africa and a major threat to the South African citrus export industry. For more than 15 years, CrleGV-SA- based biopesticides have been used as part of an integrated pest management strategy for the control of T. leucotreta in citrus orchards in South Africa, under the names Cryptogran™ and Cryptex®. While these biopesticides have been effective during this period, there are some areas in which baculovirus use could potentially be improved. Baculoviruses are notoriously slow to kill in comparison to chemical-based pesticides, and lately, pest resistance to baculoviruses has become a major concern with the development of resistance by Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) to its granulovirus occurring in the field in Europe. The consistent use of CrleGV-SA for more than 15 years in the field has raised concern that T. leucotreta could develop resistance to this virus, and has made it necessary to alter baculovirus-based management strategies to prevent this from occurring. A second baculovirus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), has recently been isolated and was shown to be effective against T. leucotreta. However, the interactions between CrleGV-SA and CrpeNPV are not yet understood and so it is important to test these interactions before both viruses are applied on the same orchards. Not only is it important to know whether these viruses could negatively impact each other, but it is also important to test whether they could interact synergistically. A synergistic interaction could not only provide a potential tool for the management of resistance, but it could also be exploited to improve baculovirus-based management of T. leucotreta. In this study, a stock of CrleGV-SA was purified by glycerol gradient centrifugation from T. leucotreta cadavers, while a stock of CrpeNPV purified from Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) cadavers was provided by River Bioscience (Pty) Ltd. These stocks were screened for purity by a multiplex polymerase chain reaction (mPCR) protocol designed to detect CrleGV-SA and CrpeNPV. The occlusion body (OB) density was then calculated using darkfield microscopy and a counting chamber. Both stocks were shown to be pure within the limits of the mPCR protocol, and the CrleGV-SA and CrpeNPV stocks were calculated to contain 3.08 × 1011 OBs/mL and 1.92 × 1011 OBs/mL respectively The first aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the dose mortality, in terms of lethal concentration. This was calculated using 7-day surface-dose biological assays for each virus and a 1:1 mixture of OBs of the two against T. leucotreta neonates. The lethal concentrations of each treatment required to kill 50 % of larvae (LC50) and 90 % of larvae (LC90) for each treatment were then calculated and compared using a probit regression. The mixed infection performed significantly better than either virus by itself, while each virus by itself did not differ significantly from the other. The LC50 for CrleGV-SA, CrpeNPV and the mixed infection were 1.53 × 104 OBs/mL, 1.15 × 104 OBs/mL and 4.38 × 103 OBs/mL respectively. The LC90 of CrleGV-SA, CrpeNPV and the mixed infection were calculated to be 4.10 × 105 OBs/mL, 1.05 × 105 OBs/mL, and 4.09 × 104 OBs/mL respectively. The second aspect of the interaction between CrleGV-SA and CrpeNPV that was investigated was the speed of kill. A time-response biological assay protocol was created that allowed for effective observation of the larvae. This was then used to generate time-mortality data that were analysed by a logit regression function to calculate and compare the treatments at the time of 50 % larval mortality (LT50) and the time of 90 % mortality (LT90). Each virus by itself did not differ significantly from the other, while the mixed infection took significantly longer to kill 50 % and 90 % of the larvae, suggesting that there is competition for resources between viruses during the secondary, systemic phase of infection. The LT50 for CrleGV-SA, CrpeNPV and the mixed infection were 117.5 hours, 113.5 hours and 139.0 hours respectively. The LT90 for CrleGV-SA, CrpeNPV and the mixed infection were 153.2 hours, 159.3, and 193.4 hours respectively. Finally, the composition of OBs recovered from the cadavers produced by the time-response biological assays were investigated by mPCR. A method for extracting gDNA from OBs in neonate-sized T. leucotreta larvae is described. The presence of CrpeNPV along with CrleGV-SA was noted in 4 out of 9 larvae inoculated with only CrleGV-SA. The presence of CrleGV-SA as well as CrpeNPV was noted in all but one larva inoculated with only CrpeNPV, and both CrleGV-SA and CrpeNPV were noted in all but one larva inoculated with a 1:1 mixture of the two, with one larva only being positive for CrleGV-SA. This suggests either stock contamination or the presence of covert infections of CrleGV-SA and CrpeNPV in the T. leucotreta population used in this study. This is the second study to report an improved lethal concentration of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates, and the first study to report the slower speed of kill of a mixed infection of CrleGV-SA and CrpeNPV against T. leucotreta neonates. While the improved lethal concentration of the mixed infection is a promising step in the future improvement of baculovirus-based biopesticides, it is at the cost of a slower speed of kill. , Thesis (MSc) -- Faculty of Science, Department of Zoology and Entomology, 2021
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Selection for improved virulence of Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) to False Codling Moth, Thaumatotibia leucotreta, by serial passage through a heterologous host

Authors: Iita, Petrus Paulus
Date: 2021-04
Subjects: Cryptophlebia leucotreta -- Biological control , Biological pest control agents , Citrus -- Diseases and pests , Baculoviruses , Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV)
Language: English
Type: thesis , text , Masters , MSc
Identifier: http://hdl.handle.net/10962/178180 , vital:42918
Description: The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is endemic to southern Africa, and strongly associated with citrus. As South African citrus production is mainly for export to foreign markets, the market access risk due to the phytosanitary status of this pest is considerable and its control is therefore imperative. Various control measures as part of a rigorous integrated pest management (IPM) programme targeted against T. leucotreta have been effective at suppressing the pest in citrus, but there is still a growing need for continued improvement of the programme and augmentation of the available control options. Of these control options, biological control, particularly the use of Cryptophlebia leucotreta granulovirus (CrleGV-SA), is a key component of IPM in citrus orchards and it has been very successful at reducing T. leucotreta populations in the field for almost two decades. There is however, a growing need for more baculovirus variants with an improved virulence against T. leucotreta for a more efficient pest management system. The newly identified insect virus, Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) offers a unique opportunity for an additional biopesticide in IPM for control of T. leucotreta in the field. This study aimed to conduct serial passaging of CrpeNPV through a heterologous host, T. leucotreta, in order to determine the potential for improved virulence or speed of kill against it. In order to select for a variant of CrpeNPV with improved virulence against T. leucotreta, a high dose (LC90) of the virus OBs was used to perform 12 serial passages through T. leucotreta larvae in surface-dose bioassays. Whole genome sequencing and analysis of the passaged virus, along with restriction endonuclease profiling in silico was performed to determine if the genetic identity of the virus had changed during serial passage, in relation to the original virus. These analyses indicated that the dominant genotype of CrpeNPV was maintained following 12 serial passages through the heterologous host. The biological activity of the passaged virus, along with the original virus was evaluated against neonate T. leucotreta in surface-dose bioassays and compared. Results from dose-response bioassays showed that the virulence of CrpeNPV did not improve after 12 serial passages. The LC50 values of the passaged virus and the original virus were estimated at 1.96 × 104 and 1.58 × 104 OBs/ml, respectively, whereas the LC90 values were estimated at 3.46 × 104 OBs/ml for the passaged virus and 3.68 × 104 for the original virus. Similarly, the results from time-response bioassays showed that the speed of kill of CrpeNPV did not improve after 12 serial passages. The LT50 values of the passaged virus and the original virus were 88.44 hours (3 days and 16 hours) and 83.74 hours (3 days and 12 hours), respectively, whereas the LT90 values were 115 hours (4 days 19 hours) for the passaged virus and 102 hours (4 days 6 hours) for the original virus. The virulence and speed of kill of the passaged virus decreased significantly, in relation to the original virus. When the full genome of the passaged virus was sequenced and analysed, only a few SNPs were detected in the viral genome, in comparison to the original virus. No detectable difference in REN digestion patterns were observed following REN analysis of gDNA of the passaged virus with several restriction enzymes in silico. The results for this study suggest that CrpeNPV may already be optimally suited to the heterologous host as it persists under these conditions without significant changes to the genome. These results have positive implications for the genetic integrity of CrpeNPV as a potential biocontrol agent in the field. This study is the first to report the virulence selection of CrpeNPV by serial passage through a heterologous host, and also the first to record bioassay data in terms of dose response (or lethal concentration) against T. leucotreta second instars. The data obtained have added to the knowledge about interactions between CrpeNPV and its heterologous host, and may be fundamental to continued investigation into the effect of serial passage on pathogenicity and genetic diversity of CrpeNPV. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2021
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Exploring the structural integrity of a picornavirus capsid

Authors: Upfold, Nicole Sarah
Date: 2020
Subjects: Picornaviruses , Immunoglobulins , Capsids (Virology) , Viruses Morphology , RNA viruses
Language: English
Type: Doctoral theses , text
Identifier: http://hdl.handle.net/10962/131837 , vital:36758 , DOI https://doi.org/10.21504/10962/131837
Description: Picornaviruses are a diverse family of small RNA viruses that cause a broad range of human and veterinary diseases. Despite decades of research into the molecular biology of these pathogens, no antivirals and few vaccines are commercially available for the treatment and prevention of picornavirus infections. The capsids of these non-enveloped viruses are involved in many important aspects of the picornavirus lifecycle, such as cell attachment and entry, uncoating, and protection of the viral RNA. Although the structures of many picornavirus capsids have been solved, a broader understanding of the molecular determinants that are required for structural integrity and stability is imperative for an improved understanding of the basic biology of these viruses, and for designing effective control strategies. Collectively, this thesis aims to elucidate the molecular determinants of structural stability and integrity in the Theiler’s murine encephalomyelitis virus capsid (TMEV). To study the TMEV GDVII capsid using biochemical techniques, neutralising polyclonal antibodies were generated against GDVII particles. The antibodies recognised linear epitopes in the C-terminus of the VP1 protein, but not those present in VP2 or VP3. The VP1 C-terminal residues were mapped to a loop above the putative receptor binding pit on the capsid surface, which prompted an investigation into the potential binding site of the TMEV co-receptor, heparan sulphate. Molecular docking revealed that heparan interacts with residues of the receptor binding pocket, as well as residues of the adjoining VP1 C-terminal loop. These findings suggest that the antibodies neutralise virus infection by preventing attachment of the virus to the co-receptor and possibly the unknown primary receptor. Few studies have identified the specific residues and interactions at subunit interfaces that significantly contribute to picornavirus capsid stability, assembly, and function. A novel in-silico screen was developed for the prediction of hotspot residues at protein-protein interfaces of a virus capsid. This screen can be applied to elucidate the residues that contribute significantly to the intraprotomer, interprotomer and interpentamer interfaces of any picornavirus capsid, on condition that the structure of the virus is available. The screen was applied to TMEV GDVII resulting in the identification of hotspots, several of which correspond to residues that are known to be important for aspects of the virus lifecycle, such as those that contribute to pH stability or form part of receptor binding sites. This observation suggests that residues involved in specific capsid functions may also play a role in capsid stability. Many of the residues identified as hotspots in TMEV corresponded to those required for assembly, uncoating, and virus growth in representative picornaviruses from various genera, suggesting that the residues that regulate capsid stability may be somewhat conserved across the family. Hotspots identified at the interpentamer interfaces of TMEV were individually substituted to alanine to further explore their importance to the TMEV lifecycle. All the amino acid substitutions prevented completion of the virus lifecycle as no CPE was observed following transfection of susceptible cells. Immunofluorescence experiments demonstrated that virus protein synthesis and RNA replication were not inhibited by substitution of the hotspot residues, but that infectivity was severely impeded. This confirmed that the residues were required for some aspect of the virus lifecycle, such as capsid assembly, or were critical for maintaining the conformational stability of the TMEV particles. Virus capsids become unstable and are prone to dissociation under certain conditions such as extreme pH and non-physiological temperatures. The thermostability of TMEV was explored by selecting GDVII virions with improved thermal tolerance through serial passage and heat exposure. Thermostable virions that could tolerate temperatures above 57 °C had reduced infective titres compared to the wild type TMEV suggesting that the virus adapted to thermal stress at the expense of viral fitness. Sequencing the capsid encoding regions of the mutant virions revealed a pair of amino acid substitutions that were present in all mutants. Additional substitutions that were unique to viruses selected at different temperatures were also identified. Most of the substitutions were located within the intraprotomer interfaces of the virus, unlike previous studies on enteroviruses where mutations were mostly localised to the receptor binding pocket. This thesis provides the first analysis of the structural determinants of TMEV capsid stability. The generation of tools to further explore the capsid structures of TMEV and other picornaviruses provides an opportunity for future studies which may contribute to the development of novel control strategies against this important family of viruses. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2020
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A computational analysis to decipher the pathways of stability, uncoating and antigenicity of human enterovirus capsids

Authors: Ross, Caroline Jane
Date: 2019
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/114788 , vital:34035 , 10.21504/10962/114788
Description: Expected release date-April 2021
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Genetic characterisation of a range of geographically distinct Helicoverpa armigera nucleopolyhedrovirus (HearNPV) isolates and evaluation of biological activity against South African populations of the African bollworm, Helicoverpa armigera (Hu bner) (Lepidoptera: Noctuidae)

Authors: Mtambanengwe, Kudzai Tapiwanashe Esau
Date: 2019
Subjects: Helicoverpa armigera -- Biological control -- South Africa , Baculoviruses -- Genetics , Agricultural pests -- Biological control -- South Africa
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/97334 , vital:31426
Description: The African bollworm, Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a pest of economic and agricultural importance globally. It is a polyphagous pest that feeds on a wide range of host plants including economically important crops. The impact it has on agricultural systems makes its control a priority. The most common method of control is using chemical pesticides; however, continuous application of the pesticides has resulted in the development of resistance. The use of biological control has been investigated and established as an effective method of control as a standalone or part of an integrated pest management (IPM) system. The use of the baculovirus Helicoverpa armigera nucleopolyhedrovirus (HearNPV), has shown promise in the control of H. armigera. Commercial formulations based on the virus are available in many global markets. However, the identification of novel HearNPV isolates will aid in the control of H. armigera as well as provide alternative isolates that may have better virulence. Three new HearNPV isolates were purified and identified from three distinct geographical South African locations H. armigera cadavers and named HearNPV-Albany, HearNPV-KZN and HearNPV-Haygrove. The genomes of two of the HearNPV isolates, namely HearNPV-Albany and HearNPV-KZN were genetically characterised and compared to other geographically distinct HearNPV isolates. Virulence studies were performed comparing the new HearNPV isolates against established commercial HearNPV formulations, Helicovir™ and Helicovex® and other geographically distinct isolated HearNPV, HearNPV-G4 and HearNPV-SP1. Two laboratory colonies were established using H. armigera collected from South African fields in the Belmont Valley near Grahamstown labelled as Albany colony and a colony provided from Haygrove Eden farm near George labelled as Haygrove colony. Biological studies were carried out using the Albany H. armigera colony comparing the rate of development, survival and fertility on bell green peppers, cabbage leaves and on artificial diet. From the biological studies, it was recorded that development and survivorship was best on artificial diet. Regular quality control was required for the maintenance of the colony and continuous generations of healthy larvae were eventually established. Diseased cadavers with signs of baculovirus infection were collected after bioprospecting from the Kwa-Zulu Natal Province in South Africa and were labelled KZN isolate; Belmont Valley near Grahamstown and were labelled Albany isolate; and Haygrove Eden farm near George and were labelled Haygrove isolate for the study. A fourth isolate made up of a crude extract of occlusion bodies (OBs) first described by Whitlock was also analysed and labelled Whitlock isolate. Occlusion bodies were extracted, purified and morphologically identified from the KZN, Albany, Haygrove and Whitlock isolates using TEM. Genomic DNA, which was extracted from the purified OBs. Using PCR, the identity of the OBs as HearNPV was confirmed. Genomic analyses were performed on HearNPV-Albany and HearNPV-KZN through genetic characterisation and comparison with other geographically distinct HearNPV genomes to confirm novelty and establish potential genetic relationships between the isolates through evolutionary distances. Full genomic sequencing of the isolated HearNPV and comparison with other geographically distinct HearNPV isolates identified genomic differences that showed that the HearNPV isolates were novel. HearNPV-Albany and HearNPV-KZN were successfully sequenced and identified as novel isolates with unique fragment patterns and unique gene sequences through deletions or insertions when compared to other geographically distinct HearNPV. This raised the potential for differences in biological activity against H. armigera larvae when tested through biological assays. HearNPV-Whit genome assembly had low quality data which resulted in many gaps and failed assembly. The biological activity of HearNPV isolates from Spain, China, South Africa and two commercial formulations were studied against the laboratory established H. armigera South African colony. The LC50 values of the different South African HearNPV isolates were established to be between 7.7 × 101 OBs.ml-1 for the most effective and 3.2 × 102 OBs.ml-1 for the least effective. The Spanish and Chinese HearNPV isolates resulted in LC50 values of 2.0 × 102 OBs.ml-1 and 1.2 × 101 OBs.ml-1 respectively. The commercial formulations resulted in the least virulence observed with an LC50 of 5.84× 102 OBs.ml-1 and 9.0 × 102 OBs.ml-1 for Helicovex® and Helicovir™ respectively. In this study, novel South African HearNPV isolates were isolated and identified. Through characterisation and bioassays against South African H. armigera populations the HearNPV isolates were shown to have different virulence in comparison to geographically distinct isolates. From this research, there is potential for development of new H. armigera biopesticides based on the novel isolates after field trial testing.
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Recovery and molecular identification of Aichi virus 1, enteric human bocaviruses and enteric human adenoviruses in untreated sewage and mussel samples collected in the Eastern Cape Province of South Africa

Authors: Onosi, Oikwathaile
Date: 2019
Subjects: Sewage -- Analysis -- South Africa -- Eastern Cape , Sewage -- Microbiology -- South Africa -- Eastern Cape , Viral pollution of water -- South Africa -- Eastern Cape , Sewage disposal in rivers, lakes, etc. -- South Africa -- Eastern Cape , Enteroviruses -- South Africa -- Eastern Cape , Picornaviruses -- South Africa -- Eastern Cape , Aichi virus 1 , Parvoviruses -- South Africa -- Eastern Cape , Adenoviruses -- South Africa -- Eastern Cape
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/69456 , vital:29539
Description: Gastroenteritis, commonly known as diarrhoeal disease, is one of the top killers responsible for substantial human morbidity and mortality especially in third world countries where most people do not have access to potable water and where hygiene levels are low. Many bacterial, viral and protozoal agents are known causes of gastroenteritis and viral gastroenteritis is responsible for over 70% of cases. Rotaviruses are the main causes of viral gastroenteritis and are responsible for most of the cases worldwide. Other viral agents associated with this disease include human noroviruses, Aichi virus 1, enteric human bocavirus, enteric human adenovirus and many other emerging viral agents such as klassivirus, Saffold virus, cosavirus and others. In 2009 the South African government introduced a rotavirus vaccine, RotaRixTM into the expanded programme on immunisation (EPI). More than a 50% decrease in diarrhoea related morbidity and mortality due to rotavirus infections was noted during surveillance studies on the efficacy of the vaccine. However, over 40% of cases of gastroenteritis are of unknown aetiology. The present study aimed to perform a preliminary study to investigate the presence of Aichi virus 1 and enteric human bocaviruses in the Eastern Cape Province by the use of molecular techniques. Furthermore, the study aimed to add to the limited molecular data about enteric adenoviruses in South Africa. Samples used in this study were swab samples collected from Belmont Valley Wastewater Treatment Plant in Grahamstown, South Africa, as well as mussel samples collected from the Swartkops River in Port Elizabeth, South Africa. Both raw sewage and shellfish give a broad idea of what microbes are circulating in the communities. In the present study, twenty swabs and twenty mussel samples were prepared by centrifugation, sonication and filtration. Samples were then subjected to transmission electron microscopy (TEM) analysis, for which the electron micrographs revealed presence of viral particles with diameters ranging from around 20 nm to just over 100 nm. Viral nucleic acids were extracted from 140 μL of the twenty swabs and twenty mussels samples using the QIAamp® Viral RNA Mini Kit, following manufacturer‟s instructions. For detection of Aichi virus 1 from the swab and mussel samples three reverse transcriptase- polymerase chain reaction (RT-PCR) assays using the Verso 1-Step RT-PCR Hot-Start Kit were developed. The first RT-PCR assay targeted amplification of the highly conserved 5′ UTR using published primers. However, despite many amplification attempts no positive results were obtained from both swab and mussel samples. It was only after the addition of DMSO (to a final concentration of 10%) that one swab sample was positive for this assay. In addition, a 2-step RT-PCR was developed using the Maxima H Minus First Strand cDNA Synthesis Kit. By using this 2-step RT-PCR assay, an additional swab sample was positive for the Aichi virus 1 5′ UTR. Using Basic Logarithm Alignment Search Tool (BLAST) analysis these two samples were 98% identical to an Aichi virus isolate from South Korea. The second one-step RT-PCR assay targeted amplification of the 266 bp partial 3CD coding region of Aichi virus 1 using published primers. By using this assay, positive results were obtained from both the swab and mussel samples, which when analysed by BLAST were all 99% identical to various Aichi virus 1 isolates in GenBank. A phylogenetic tree constructed based on this region showed that isolates from the present study clustered with Genotype B isolates in GenBank. The third assay was a semi-nested RT-PCR assay that targeted amplification of the hypervariable VP1 coding region of Aichi virus 1 using a combination of published primers and those designed in the present study. Amplicons which were 472 bp in size were produced from two swab samples. When analysed by BLAST, these two swab samples had percentage identities of 98% to an Aichi virus isolate from South Korea. A phylogenetic tree constructed based on this region showed that isolates from the present study clustered with Genotype B isolates in GenBank. This was consistent with phylogenetic results discussed above which were based on the partial 3CD region. For detection of enteric human bocaviruses from the swab and mussel samples a nested polymerase chain reaction (PCR) assay, using the Ampliqon Taq PCR kit (Ampliqon Bio Reagents and Molecular Diagnostics, Denmark) was developed based on PCR amplification of the 382 bp partial VP1/VP2 coding region using published primers. A total of six swab samples and six mussel samples were analysed for which five swabs and six mussel samples gave positive results. When analysed by BLAST, the swab samples had percentage identities of between 98% and 99% to an enteric human bocavirus 3 strain from China while the mussel samples were all 99% identical to an enteric human bocavirus 2 isolate from Australia. A phylogenetic tree constructed based on this VP1/VP2 region showed that isolates from the present study clustered with human bocavirus 2 and human bocavirus 3 isolates in GenBank for those isolated from swab samples and mussel samples respectively. Lastly, for detection of enteric human adenoviruses from the swab and mussel samples a nested PCR assay, using the Ampliqon Taq PCR kit (Ampliqon Bio Reagents and Molecular Diagnostics, Denmark) was developed. This reaction was based on PCR amplification of the 168 bp partial hexon coding region using published primers for which ten swab samples gave positive results. When analysed by BLAST, the swab samples had percentage identities of between 96% and 99% to enteric human adenoviruses in GenBank. A phylogenetic tree constructed based on the hexon coding region showed that isolates from the present study clustered with subtypes C, D and F which are associated with gastroenteritis worldwide. Despite several amplification attempts no positive results were obtained from mussel samples. The results from the present study show that Aichi virus 1, enteric bocaviruses and enteric adenoviruses are present in the Eastern Cape Province of South Africa. These viruses could possibly be responsible for enteric infections in South Africa. Although only a few samples were analysed, this study is the first to confirm the presence of Aichi virus 1 and enteric bocaviruses in South Africa and provides a platform for further investigation into prevalence and epidemiology of these viruses in the country.
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Baculovirus synergism: investigating mixed alphabaculovirus and betabaculovirus infections in the false codling moth, thaumatotibia leucotreta, for improved pest control

Authors: Jukes, Michael David
Date: 2018
Subjects: Baculoviruses , Cryptophlebia leucotreta -- Biological control , Citrus -- Diseases and pests -- South Africa , Pests -- Integrated control , Nucleopolyhedroviruses , Natural pesticides , Cryptophlebia leucotreta granulovirus (CrleGV)
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/61797 , vital:28061
Description: Baculovirus based biopesticides are an effective and environmentally friendly approach for the control of agriculturally important insect pests. The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is indigenous to southern Africa and is a major pest of citrus crops. This moth poses a serious risk to export of fruit to foreign markets and the control of this pest is therefore imperative. The Cryptophlebia leucotreta granulovirus (CrleGV) has been commercially formulated into the products Cryptogran™ and Cryptex®. These products have been used successfully for over a decade as part of a rigorous integrated pest management (IPM) programme to control T. leucotreta in South Africa. There is however, a continuous need to improve this programme while also addressing new challenges as they arise. An example of a rising concern is the possibility of resistance developing towards CrleGV. This was seen in Europe with field populations of the codling moth, Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae), which developed resistance to the Mexican isolate of the Cydia pomonella granulovirus (CpGV-M). To prevent such a scenario occurring in South Africa, there is a need to improve existing methods of control. For example, additional baculovirus variants can be isolated and characterised for determining virulence, which can then be developed as new biopesticides. Additionally, the potential for synergistic effects between different baculoviruses infecting the same host can be explored for improved virulence. A novel nucleopolyhedrovirus was recently identified in T. leucotreta larval homogenates which were also infected with CrleGV. This provided unique opportunities for continued research and development. In this study, a method using C. pomonella larvae, which can be infected by the NPV but not by CrleGV, was developed to separate the NPV from GV-NPV mixtures in an in vivo system. Examination of NPV OBs by transmission electron microscopy showed purified occlusion bodies with a single nucleopolyhedrovirus morphology (SNPV). Genetic characterisation identified the novel NPV as Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV), which was recently isolated from the litchi moth, Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae). To begin examining the potential for synergism between the two viruses, a multiplex PCR assay was developed to accurately detect CrleGV and/or CrpeNPV in mixed infections. This assay was applied to various samples to screen for the presence of CrpeNPV and CrleGV. Additionally, a validation experiment was performed using different combinations of CrpeNPV and/or CrleGV to evaluate the effectiveness of the mPCR assay. The results obtained indicated a high degree of specificity with the correct amplicons generated for each test sample. The biological activity of CrpeNPV and CrleGV were evaluated using surface dose bioassays, both individually and in various combinations, against T. leucotreta neonate larvae in a laboratory setting. A synergistic effect was recorded in the combination treatments, showing improved virulence when compared against each virus in isolation. The LC90 for CrpeNPV and CrleGV when applied alone against T. leucotreta was calculated to be 2.75*106 and 3.00*106 OBs.ml"1 respectively. These values decreased to 1.07*106 and 7.18*105 OBs.ml"1 when combinations of CrleGV and CrpeNPV were applied at ratios of 3:1 and 1:3 respectively. These results indicate a potential for developing improved biopesticides for the control of T. leucotreta in the field. To better understand the interactions between CrleGV and CrpeNPV, experiments involving the serial passage of these viruses through T. leucotreta larvae were performed. This was done using each virus in isolation as well as both viruses in different combinations. Genomic DNA was extracted from recovered occlusion bodies after each passage and examined by multiplex and quantitative PCR. This analysis enabled the detection of each virus present throughout this assay, as well as recording shifts in the ratio of CrleGV and CrpeNPV at each passage. CrleGV rapidly became the dominant virus in all treatments, indicating a potentially antagonistic interaction during serial passage. Additionally, CrpeNPV and CrleGV were detected in treatments which were not originally inoculated with one or either virus, indicating potential covert infections in T. leucotreta. Occlusion bodies recovered from the final passage were used to inoculate C. pomonella larvae to isolate CrpeNPV from CrleGV. Genomic DNA was extracted from these CrpeNPV OBs and examined by restriction endonuclease assays and next generation sequencing. This enabled the identification of potential recombination events which may have occurred during the dual GV and NPV infections throughout the passage assay. No recombination events were identified in the CrpeNPV genome sequences assembled from virus collected at the end of the passage assay. Lastly, the efficacy of CrpeNPV and CrleGV, both alone and in various combinations, was evaluated in the field. In two separate trials conducted on citrus, unfavorable field conditions resulted in no significant reduction in fruit infestation for both the virus and chemical treatments. While not statistically significant, virus treatments were recorded to have the lowest levels of fruit infestation with a measured reduction of up to 64 %. This study is the first to report a synergistic effect between CrleGV and CrpeNPV in T. leucotreta. The discovery of beneficial interactions creates an opportunity for the development of novel biopesticides for improved control of this pest in South Africa.
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Isolation, identification and genetic characterisation of a microsporidium isolated from the carob moth, Ectomyelois ceratoniae (Lepidoptera: Pyralidae)

Authors: Lloyd, Melissa
Date: 2018
Subjects: Pyralidae , Pyralidae -- Genetics , Pyralidae -- Phylogeny , Pyralidae -- Pathogens , Cladistic analysis , Transmission electron microscopy , Carob moth (Ectomyelois ceratoniae)
Language: English
Type: text , Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/61894 , vital:28075
Description: Carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) is an economically important pest, yet its biology and pest status on citrus in South Africa was, until recently, poorly understood. A study was initiated to determine the cause of collapse of a laboratory carob moth colony that was established to investigate the biology of carob moth on citrus and to develop integrated management strategies for the pest. An organism was isolated from deceased larvae and was morphologically identified as a microsporidium, based on transmission electron microscopy. Microsporidia are obligate intracellular parasites that have been found to infect almost all eukaryotes. Several Nosema species have been isolated from economically important insect pests, yet little genetic information is available from online databases for identification. Mature spores were recovered and measured using transmission electron microscopy. Spores were ovocylindrical with a wrinkled exospore, and had a length of 2.8 ± 0.02 pm and a width of 1.6 ± 0.04 pm. The identity of the microsporidium was confirmed by PCR amplification, sequencing and analysis of the regions encoding the ribosomal RNA. BLAST analysis of the different rRNA regions amplified showed that the microsporidium shared a 96 - 99 % identity with Nosema sp. M-Pr, Nosema carpocapsae, Nosema oulemae, Nosema sp. CO1, Microsporidium 57864, and Nosema bombi. Phylogenetic analysis of the SSU and LSU rRNA genes showed that the microsporidium clustered with the Nosema / Vairimorpha clade, supported by a bootstrap value of 100. The organisation of the RNA cistron was determined by PCR amplification using the primer set 18f and L1328r to be 5’-SSU-ITS-LSU-IGS-5S-3’, which confirms the placement of the microsporidium within the Nosema / Vairimorpha clade. Because the BLAST results showed a close relationship with Nosema carpocapsae, a microsporidium infecting codling moth, the pathogenicity of the microsporidium was tested against codling moth by inoculating artificial diet with a high spore concentration of 1.1 x 107 spores/ml and a low spore concentration of 1.1 x 104 spores/ml. DNA was extracted from deceased larvae inoculated with the high concentration, and PCR of the SSU rRNA gene and bacterial 16S region was performed. Mortality in the high concentration experiment was significant (p = 0.05), but the cause of infection was determined to be a bacterium, through sequencing and BLAST analysis of the bacterial 16S rDNA. The bacterium shared a 99 % identity with Bacillus cereus. Percentage mortality (p = 0.09), larval mass (p = 0.09) and instar (p = 0.24) did not differ significantly between treatments in the low concentration experiment. DNA was extracted from the larvae and PCR amplification of the SSU rRNA gene was performed to determine whether microsporidia were present. No SSU bands were observed in any of the treatments and percentage mortality was not significant, thus it was determined that no infection occurred. This is the first study to report the genetic characterisation of a microsporidium isolated from carob moth and provides important genetic information for classification of microsporidia within the Nosema / Vairimorpha clade. It is also one of few studies in which the complete rRNA cistron of a species within the Nosema / Vairimorpha clade has been sequenced. The identification of a microsporidium from a laboratory colony of carob moth is important as it provides information about pathogens infecting the carob moth and constraints to carob moth rearing, which is useful for further studies on rearing carob moth and for establishment of a clean colony for research purposes.
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Yeast-baculovirus synergism: investigating mixed infections for improved management of the false codling moth, Thaumatotibia leucotreta

Authors: Van der Merwe, Marcél
Date: 2018
Subjects: Cryptophlebia leucotreta , Baculoviruses , Yeast , Citrus Diseases and pests , Biological pest control agents , Pests Integrated control
Language: English
Type: Master's theses , text
Identifier: http://hdl.handle.net/10962/62963 , vital:28347
Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) or otherwise commonly known as the false codling moth is an indigenous pest of the citrus industry in southern Africa. The pest is highly significant as it impacts negatively on the export of fresh citrus fruits from South Africa to international markets. To control T. leucotreta in South Africa, an integrated pest management (IPM) programme has been implemented. One component of this programme is the baculovirus Cryptophlebia leucotreta granulovirus (CrleGV-SA) which has been formulated into the products Cryptogran™ and Cryptex®. It has previously been reported that there is a mutualistic association between Cydia pomonella (L.) (Lepidoptera: Tortricidae) also known as codling moth, and epiphytic yeasts. Cydia pomonella larval feeding galleries were colonised by yeasts and this, in turn, reduced larval mortality and enhanced larval development. It has been demonstrated in laboratory assays and field trials that combining yeast and brown cane sugar with Cydia pomonella granulovirus (CpGV) significantly increased larval mortality and lowered the proportion of injured apple fruit. This suggests that yeasts can enhance the effectiveness of an insect virus in managing pest larvae. In this study, we proposed to determine which species of yeast occur naturally in the digestive tract, frass and on the epidermis of T. leucotreta larvae and to examine whether any of these yeasts, when combined with the CrleGV-SA, have a synergistic effect in increasing mortality of T. leucotreta larvae. Firstly, Navel oranges infested with T. leucotreta larvae were collected from orchards in Sundays River Valley in Eastern Cape of South Africa. Larvae were extracted and analysed for the presence of yeast on their surface, or in their gut and frass. Four yeasts were isolated from T. leucotreta larvae and identified down to species level via PCR amplification and sequencing of internal transcribed spacer (ITS) region and D1/D2 domain of the large subunit (LSU) of rDNA region. These yeasts were isolated from the frass, epidermis and digestive tract of T. leucotreta larvae. The yeast isolates were identified as Meyerozyma caribbica, Pichia kluyveri, Pichia kudriavzevii and Hanseniaspora opuntiae. A yeast preference assay was conducted on female T. leucotreta moths to examine whether any of the isolated yeast species affected their oviposition preference. Navel oranges were inoculated with the isolated yeast species at a concentration of 6 × 108 cells.ml-1. The assay also included a Brewer’s yeast and distilled water control. Pichia kudriavzevii was shown to be the preferred yeast species for oviposition, as significantly more eggs were deposited on Navel oranges inoculated with this yeast compared to the other treatments. Lastly, a detached fruit bioassay was performed to evaluate the efficacy of mixing P. kudriavzevii with CrleGV-SA to enhance T. leucotreta larvae mortality. Pichia kudriavzevii was selected as it was demonstrated as having an effect on the oviposition preference of female T. leucotreta moths. The concentration at which P. kudriavzevii was applied remained the same as in the preference assay while CrleGV-SA was applied at lethal concentration required to kill 50 % of the population (9.31 × 107 OBs.ml-1). Although an increase in larval mortality was observed between CrleGV-SA being applied alone and the yeast/virus mixture, this result was determined not to be statistically significant. The experiments performed in this study provide a platform for further research into the application of a yeast-virus combination as a novel control option for T. leucotreta in the field. , Thesis (MSc) -- Faculty of Science, Biochemistry and Microbiology, 2018
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The development of techniques for the identification of novel viruses associated with acute infantile gastroenteritis in South Africa

Authors: Jaquet, Brittany J
Date: 2017
Subjects: Gastroenteritis in children -- Treatment , Gastroenteritis in children -- Treatment -- South Africa , Antiviral agents , Viral vaccines , Rotaviruses , Virus diseases in children
Language: English
Type: Thesis , Masters , MSc
Identifier: http://hdl.handle.net/10962/38013 , vital:24725
Description: Gastroenteritis is a serious disease affecting both children and adults globally, but is more predominant in children with over half a million deaths reported each year. The leading cause of this disease is rotavirus, which accounts for 38% of all hospitalised cases. There has, however, been a significant decrease in the number of deaths associated with rotavirus worldwide since the introduction of the two vaccines, Rotarix® and RotaTeq®. A large number of cases are therefore either associated with other viruses, such as norovirus, Aichi virus (AiV) or Saffold virus (SAFV), or are of unknown aetiology. This study thus aims to develop techniques for the identification of viruses associated with gastroenteritis. Theiler’s murine encephalomyelitis virus (TMEV) was used to develop the sample preparation, transmission electron microscopy and RT-PCR techniques used in this study. This virus was chosen as a replication system using baby hamster kidney cells can be used to create high concentrations of viral particles from which RNA can be extracted preventing the waste of the limited samples. The virus particles are also similar in size and morphology to the viruses to be identified in this study, as it belongs to the same family. After sample preparation, TEM analysis showed the presence of small, round, non-enveloped virus particles in the TMEV sample. Due to the low concentration of virus particles, PEG precipitation was performed using both 0.15 M and 0.25 M NaCl and 8% (w/v) PEG 6000. TEM analysis then showed an increase in viral particle concentration, with the highest concentration observed at 0.25 M NaCl and 8% PEG 6000. RNA was successfully extracted and RT-PCR assays were performed for both the VP1 and 2B coding regions of TMEV. A method for creating a positive control for the RT-PCR assay was developed by the in vitro transcription of RNA from pTMEV, which contains the cDNA of TMEV. The RNA was then used as the template for the 2B two-step RT-PCR assay. A product of 412 bp was successfully amplified from the in vitro transcribed RNA and the sensitivity of the RT-PCR assay was determined. Using a Norovirus GII positive stool sample provided by Maureen Taylor, a nested RT-PCR assay was developed for the NoV GII N/S domain using a previously-published primer set and cycling parameters. A 342 bp product was successfully amplified from the RNA extracted from the stool sample and cloned into pGEM®-T Easy to produce pNoVGII. Using the plasmid containing the AiV 5’UTR and the PCR amplicon for AiV 3CD, RT-PCR assays were developed for AiV 5’UTR and partial 3CD. The RT-PCR assays produced a 1008 bp product for AiV 5’UTR and 266 bp for AiV 3CD, which were cloned into pGEM®-T Easy to produce pAiV5’UTR and pAiV3CD, respectively. Using in vitro transcribed RNA from pNoVGII, pAiV5’UTR, pAiV3CD and pSAFV, which contains the SAFV cDNA, positive controls were developed for the RT-PCR assays for NoV GII, AiV 5’UTR, AiV 3CD and SAFV 2C. The sensitivity of these assays was determined. The samples chosen for this study include wastewater collected from the Belmont Valley water treatment plant, oysters suspected to be infected with viruses collected from Port Elizabeth, South Africa and 30 stool samples from symptomatic patients. With the methods developed using TMEV, the wastewater, oysters and 30 stool samples were filter-sterilised, concentrated and screened by TEM. All samples showed the presence of virus particles. RNA was successfully extracted and the wastewater, oyster and 30 stool samples were screened for NoV GII using the NoV GII RT-PCR assay. The wastewater, oysters and 11 of the stool samples produced the 342 bp NoV GII PCR product and BLAST analysis determined the nucleotide sequences to be NoV GII.4. This shows that this study was able to develop sample preparation techniques and TEM analysis for selected samples and RT-PCR assays for NoV GII, AiV and SAFV. The NoV GII RT-PCR assay was successfully used for the screening of the wastewater, oysters and 30 stool samples for NoV GII. Due to the high number of gastroenteritis cases with unknown aetiology in South Africa, the development of techniques for the identification of NoV, AiV, SAFV and other viruses is very important. The identification of these viruses will allow for better surveillance, treatment and prevention of gastroenteritis in South Africa.
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The isolation and genetic characterisation of a novel alphabaculovirus for the microbial control of Cryptophlebia peltastica and closely related tortricid pests

Authors: Marsberg, Tamryn
Date: 2017
Language: English
Type: text , Thesis , Doctoral , PhD
Identifier: http://hdl.handle.net/10962/59292 , vital:27543
Description: Cryptophlebia peltastica (Meyrick) (Lepidoptera: Tortricidae) is an economically damaging pest of litchis and macadamias in South Africa. Cryptophlebia peltastica causes both pre- and post-harvest damage to litchis, reducing overall yields and thus classifying the pest as a phytosanitary risk. Various control methods have been implemented against C. peltastica in an integrated pest management programme. These control methods include chemical control, cultural control and biological control. However, these methods have not yet provided satisfactory control as of yet. As a result, an alternative control option needs to be identified and implemented into the IPM programme. An alternative method of control that has proved successful in other agricultural sectors and not yet implemented in the control of C. peltastica is that of microbial control, specifically the use of baculovirus biopesticides. This study aimed to isolate and characterise a novel baculovirus from a laboratory culture of C. peltastica that could be used as a commercially available baculovirus biopesticide. In order to isolate a baculovirus a laboratory culture of C. peltastica was successfully established at Rhodes University, Grahamstown, South Africa. This is the first time a laboratory culture of C. peltastica has been established. This allowed for various biological aspects of the pest to be determined, which included: length of the life cycle, fecundity and time to oviposition, egg and larval development and percentage hatch. The results obtained from these studies found that the biology of C. peltastica was similar to that of Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae). Once the laboratory culture had reached high densities, larvae showing symptoms of baculovirus infection were observed. Symptomatic larvae were collected and examined for the presence of a baculovirus. An alphabaculovirus (NPV) was successfully isolated and morphologically identified using purified OBs that were sectioned and observed by transmission electron microscopy. This was then confirmed by amplifying the polyhedrin gene region using degenerate primers. A BLAST analysis found a 93% similarity to a partial polyhedrin gene sequence to be that of Epinotia granitalis (Butler) (Lepidoptera: Tortricidae). The alphabaculovirus was then genetically characterised by generating restriction profiles and sequencing the whole genome. Due to the novelty of the virus, no comparison could be made. The biological activity of the alphabaculovirus was then tested against C. peltastica and two closely related Tortricidae pests: T. leucotreta and Cydiapomonella (Linnaeous) (Lepidoptera: Tortricidae). The alphabaculovirus was highly virulent against all three species. The lethal concentrations (LC50 and LC90) for the virus against C. peltastica was 8.19 x 103 and 3.33 x 105 OBs/ml. The LC50 and LC90 for T. leucotreta was 2.29 x 103 and 9.97 x 104 OBs/ml, respectively and C. pomonella had a LC50 of 1.43 x 103 OBs/ml and LC90 1.26 x 104 OBs/ml. The virus was particularly virulent against T. leucotreta and C. pomonella as compared to C. peltastica. The biological activity of the alphabaculovirus was also tested against CpGV resistant European C. pomonella. From the results it was observed that the virus had the ability to overcome the resistance in C. pomonella and could potentially be used in the resistance management of C. pomonella. With the successful biological activity results obtained from this study, preliminary investigation were made into the mass production of the alphabaculovirus using both the in vivo and in vitro production methods. For in vivo production both the homologous host (C. peltastica) and a heterologous host (T leucotreta) were investigated. Preliminary studies focused on determining the biological activity in fifth instars of both hosts. Fifth instar LC50 and LC90 values for C. peltastica were 3.43 x 103 and 1.11 x 107 OBs/ml and for T. leucotreta the LC50 and LC90 values were 2.53 x 103 and 8.82 x 106 OBs/ml, respectively. The average yield of virus produced in each species was also determined. Cryptophlebia peltastica had the highest viral yield of 5.37 x 1010 OBs/larva and 2.93 x 1010 OBs/larva for T. leucotreta. The results obtained, from the preliminary investigation concluded that the virus could be produced in vivo in both C. peltastica and T. leucotreta, however further research is required into the mass production in both hosts. The in vitro production of the virus was also considered and the susceptibility of the virus was tested against the C. pomonella cell line, Cp14R. After infection of the Cp14R cells with budded virus collected from fifth instar C. peltastica larvae, OBs could be observed after three days. Thus, the alphabaculovirus is susceptible to the Cp14R cell line, thus has the potential to be produced in vitro and further characterised. This study is the first to report of the identification and characterisation of a novel alphabaculovirus isolated from a laboratory reared culture of C. peltastica and the potential for it to be commercially developed into a bipoesticide and used against Tortricidae pests.
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Genetic and biological characterisation of a novel South African Cydia pomonella granulovirus (CpGV-SA) isolate

Authors: Motsoeneng, Boitumelo Madika
Date: 2016
Language: English
Type: Thesis , Masters , MSc
Identifier: vital:20503 , http://hdl.handle.net/10962/d1021266
Description: The codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae), is the primary pest of pome fruit cultivated worldwide. The control of this insect pest has been dependent on the frequent use of broad-spectrum chemical pesticides, which has led to the development of resistance in pest populations and negative effects on human health and the environment. The Betabaculovirus of C. pomonella has successfully been applied as a biological control agent in integrated pest management (IPM) programmes for the suppression of pest populations worldwide. Previously, all Cydia pomonella granulovirus (CpGV) biopesticides were based on a Mexican isolate (CpGV-M) and although these products are highly efficient at controlling C. pomonella, resistance cases have been reported across Europe. The identification of novel CpGV isolates as additional or alternative control agents to manage resistance is therefore necessary. This study aimed to genetically and biologically characterise a novel South African C. pomonella granulovirus isolate and to test its virulence against neonate larvae. Based on the morphology of the occlusion bodies observed using transmission electron microscopy, granuloviruses were recovered from diseased and dead larvae collected from an orchard in South Africa where no virus applications had been made. DNA was extracted and the identification of the isolated granulovirus was achieved through the PCR amplification and sequencing of the lef-8, lef-9, granulin and egt genes. Submission of the gene sequences to BLAST revealed high percentage identities to sequences from various CpGV isolates, resulting in the naming of the isolate in this study as the South African Cydia pomonella granulovirus (CpGV-SA) isolate. Phylogenetic analysis based on the single nucleotide polymorphisms (SNPs) detected in the lef-8, lef-9 and granulin nucleotide sequences grouped the South African isolate with CpGV-E2 (genome type B) and CpGV-S (genome type E). The CpGV-SA isolate was further genetically characterised by restriction endonuclease analysis and complete sequencing of the genomic DNA. Differences were observed for the BamHI, EcoRI, PstI and XhoI profiles of CpGV-SA in comparison to the respective profiles generated for CpGV-M extracted from a biopesticide, Carpovirusine® (Arysta Lifescience, France). Several genetic variations between the complete genome sequence of CpGV-SA and the reference isolate, CpGV-M1, as well as a recent genome submission of CpGV-M, both representing genome type A were observed. The complete genome analysis confirmed that CpGV-SA is genetically different from the Mexican CpGV isolate, used in thedevelopment of most biopesticides. In silico restriction profiles of the genome sequence obtained for CpGV-SA and genome sequences of genetically different CpGV isolates originating from Mexico (M1 and M), England (E2), Canada (S) and Iran (I12 and I07), available on the NCBI’s GenBank database confirmed that CpGV-SA is of mixed genotypes. Furthermore, the South African isolate shared the single common difference found in the pe38 gene of resistance overcoming isolates, which was the absence of an internal 24 nucleotide repeat present in CpGV-M1. In addition to the common difference, SNPs detected in the pe38 gene grouped the isolate with the CpGV-S isolate, suggesting that the CpGV-SA isolate is predominantly of genome type E. To determine the biological activity of CpGV-SA against neonate C. pomonella larvae, surface bioassays were conducted alongside CpGV-M (Carpovirusine®) bioassays. The LC50 and LC90 values for the South African isolate were 1.6 × 103 and 1.2 × 105 OBs/ml respectively. The LT50 was determined to be 135 hours. These values were similar to the values obtained for CpGV-M (Carpovirusine®). The results in this study suggest that a novel South African CpGV isolate of mixed genotypes, potentially able to overcome resistance in C. pomonella, with biological activity similar to CpGV-M (Carpovirusine®) and important for the control of C. pomonella was recovered. The CpGV-SA isolate could therefore potentially be developed into a biopesticide for use in resistance management strategies against C. pomonella populations in South Africa.
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Prediction of interacting motifs within the protein subunits of Picornavirus capsids

Authors: Ross, Caroline Jane
Date: 2015
Subjects: Picornaviruses , Antiviral agents , Poliovirus , Coxsackieviruses , Hepatitis A virus , Foot-and-mouth disease virus , Viral proteins
Language: English
Type: Thesis , Masters , MSc
Identifier: vital:4151 , http://hdl.handle.net/10962/d1017912
Description: The Picornaviridae family contains a number of pathogens which are economically important including Poliovirus, Coxsakievirus, Hepatitis A Virus, and Foot-and-Mouth-Disease-Virus. Recently the emergence of novel picornaviruses associated with gastrointestinal, neurological and respiratory diseases in humans has been reported. Although effective vaccines for viruses such as FMDV, PV and HAV have been developed there are currently no antivirals available for the treatment of picornavirus infections. Picornaviruses proteins are classified as: the structural proteins VP1, VP2, VP3 and VP4 which form the subunits of the viral capsid and the replication proteins which function as proteases, RNA-polymerases, primers and membrane binding proteins. Although the host specificity and viral pathogenicity varies across members of the family, the icosahedral capsid is highly conserved. The capsid consists of 60 protomers, each containing a single copy of VP1, VP2 and VP3. A fourth capsid protein, VP4, resides on the internal side of the capsid. Capsid assembly is integral to life-cycle of picornaviruses; however the process is complex and not fully-understood. The overall aim of the study was to broaden the understanding of the evolution and function of the structural proteins across the Picornaviridae family. Firstly a comprehensive analysis of the phylogenetic relationships amongst the individual structural proteins was performed. The functions of the structural proteins were further investigated by an exhaustive motif analysis. A subsequent structural analysis of highly conserved motifs was performed with respect to representative enteroviruses, Foot-and-Mouth-Disease-Virus and Theiler’s Virus. This was supplemented by the in silico prediction of interacting residues within the crystal structures of these protomers. Findings in this study suggest that the capsid proteins may be evolving independently from the replication proteins through possible inter-typic recombination of functional protein regions. Moreover the study predicts that protomer assembly may be facilitated through a network of multiple subunit-subunit interactions. Multiple conserved motifs and principle residues predicted to facilitate capsid subunit-subunit interactions were identified. It was also concluded that motif conservation may support the theory of inter-typic recombination between closely related virus sub-types. As capsid assembly is critical to the viral life-cycle, the principle interacting motifs may serve as novel drug targets for the antiviral treatment of picornavirus infections. Thus the findings in the study may be fundamental to the development of treatments which are more economically feasible or clinically effective than current vaccinations.
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Production of Cydia pomonella granulovirus (CpGV) in a heteralogous host, Thaumatotibia Leucotreta (Meyrick) (False codling moth)

Authors: Chambers, Craig Brian
Date: 2015
Subjects: Cryptophlebia leucotreta -- South Africa , Codling moth -- South Africa , Apples -- Diseases and pests -- South Africa , Codling moth -- Biological control -- South Africa , Insect pests -- Biological control -- South Africa , Biological pest control agents -- South Africa , Baculoviruses -- South Africa
Language: English
Type: Thesis , Doctoral , PhD
Identifier: vital:5935 , http://hdl.handle.net/10962/d1017906
Description: Cydia pomonella (Linnaeus) (Family: Tortricidae), the codling moth, is considered one of the most significant pests of apples and pears worldwide, causing up to 80% crop loss in orchards if no control measures are applied. Cydia pomonella is oligophagous feeding on a number of alternate hosts including quince, walnuts, apricots, peaches, plums and nectarines. Historically the control of this pest has been achieved with the use of various chemical control strategies which have maintained pest levels below the economic threshold at a relatively low cost to the grower. However, there are serious concerns surrounding the use of chemical insecticides including the development of resistance in insect populations, the banning of various insecticides, regulations for lowering of the maximum residue level and employee and consumer safety. For this reason, alternate measures of control are slowly being adopted by growers such as mating disruption, cultural methods and the use of baculovirus biopesticides as part of integrated pest management programmes. The reluctance of growers to accept baculovirus or other biological control products in the past has been due to questionable product quality and inconsistencies in their field performance. Moreover, the development and application of biological control products is more costly than the use of chemical alternatives. Baculoviruses are arthropod specific viruses that are highly virulent to a number of lepidopteran species. Due to the virulence and host specificity of baculoviruses, Cydia pomonella granulovirus has been extensively and successfully used as part of integrated pest management systems for the control of C. pomonella in Europe and around the world, including South Africa. Commercial formulations have been typically based on the Mexican strain of CpGV. However due to long-term multiple applications of CpGV and the reliance on CpGV in organic farming practices in Europe, resistance to the CpGV-M strain has developed in a number of field populations of C. pomonella. This study aimed to identify and characterize novel isolates of CpGV in South Africa and compare their virulence with the commercial standard CpGV-M. Secondly, since C. pomonella is difficult to culture on a large scale, an alternate method of CpGV production was investigated in order to determine if CpGV could be produced more efficiently and at a reduced cost without negatively impacting the quality of the product. Several isolates of CpGV were recovered either from field collected larvae or from a laboratory-reared C. pomonella colony. Characterisation of DNA profiles using a variety of restriction enzymes revealed that only a single isolate, CpGV-SA, was genetically different from the Mexican strain of the virus used in the commercially available CpGV based products in South Africa. In dose-response bioassays using CpGV-SA, LC₅₀ and LC₉₀ values for neonate C. pomonella larvae were 3.18 x 10³ OBs/ml and 7.33 x 10⁴ respectively. A comparison of these values with those of CpGV-M indicated no significant difference in the virulence of the two isolates under laboratory conditions. This is a first report of a genetically distinct CpGV isolate in South Africa. The biological activity and novelty of CpGV-SA makes this isolate a potentially important tool for CpGV resistance management in South Africa. In order to justify production of CpGV in an alternative host, studies on the comparative biological performance of C. pomonella and T. leucotreta based on oviposition, time to hatch, larval developmental times and rearing efficiency as well as production costs were performed. Thaumatotibia leucotreta was found to be more fecund and to have significantly shorter egg and larval developmental times. In addition, larval production per unit of artificial diet was significantly higher than for C. pomonella. This resulted in T. leucotreta being more cost effective to produce with implications for reduced insectary space, sanitation practices as well as the labour component of production. Virus yield data generated by inoculation both C. pomonella and T. leucotreta with nine concentrations of CpGV resulted in comparable virus yields, justifying the continuation of the research into production of CpGV in T. leucotreta. It was important to determine the LC and LT values required for mass production of CpGV in late instar T. leucotreta larvae. Dose- and time-response bioassays with CpGV-M were conducted on artificial diet to determine these values. Fourth instar LC₅₀ and LC₉₀ values were 5.96 x 10³ OBs/ml and 1.64 x 10⁵ OBs/ml respectively. LT50 and LT90 values were 81.10 hours and 88.58 hours respectively. Fifth instar LC₅₀ and LC₉₀ values were 6.88 x 10⁴ OBs/ml and 9.78 x 10⁶ OBs/ml respectively. LT₅₀ and LT₉₀ values were 111.56 hours and 137.57 hours respectively. Virus produced in fourth instar T. leucotreta larvae was bioassayed against C. pomonella neonate larvae and compared to CpGV-M to establish if production in the heterologous host negatively affected the virulence of the isolate. No significant difference in virulence was observed between virus produced in T. leucotreta and that produced in C. pomonella. The data generated in the bioassays was used in CpGV mass production trials to evaluate production. All production methods tested produced acceptable virus yields. To examine the quality of the virus product, genomic DNA was extracted from larval cadavers and subjected to REN analysis with HindIII. The resulting DNA profiles indicated that the virus product was contaminated with the homologous virus, CrleGV. Based on the above results, the use of T. leucotreta as an alternate host for the in vivo production of CpGV on a commercial basis is not at this stage viable and requires further investigation before this production methodology can be reliable used to produce CpGV. However, this study has shown that CpGV can be produced in a homologous host, T. leucotreta and significant strides have been made towards developing a set of quality control standards that are essential for further development of successful production methodology. Finally a novel isolate of CpGV has been identified with comparable virulence to the CpGV-M. This is an important finding as it has broad reaching implications for resistance management of CpGV products in South Africa.
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The isolation, genetic characterisation and biological activity of a South African Phthorimaea operculella granulovirus (PhopGV-SA) for the control of the Potato Tuber Moth, Phthorimaea operculella (Zeller)

Authors: Jukes, Michael David
Date: 2015
Subjects: Potato tuberworm , Potatoes -- Diseases and pests -- South Africa , Baculoviruses , Natural pesticides , Biological pest control agents , Potato tuberworm -- Biological control , Restriction enzymes, DNA
Language: English
Type: Thesis , Masters , MSc
Identifier: vital:4147 , http://hdl.handle.net/10962/d1017908
Description: The potato tuber moth, Phthorimaea operculella (Zeller), is a major pest of potato crops worldwide causing significant damage to both field and stored tubers. The current control method in South Africa involves chemical insecticides, however, there is growing concern on the health and environmental risks of their use. The development of novel biopesticide based control methods may offer a potential solution for the future of insecticides. In this study a baculovirus was successfully isolated from a laboratory population of P. operculella. Transmission electron micrographs revealed granulovirus-like particles. DNA was extracted from recovered occlusion bodies and used for the PCR amplification of the lef-8, lef-9, granulin and egt genes. Sequence data was obtained and submitted to BLAST identifying the virus as a South African isolate of Phthorimaea operculella granulovirus (PhopGV-SA). Phylogenetic analysis of the lef-8, lef-9 and granulin amino acid sequences grouped the South African isolate with PhopGV-1346. Comparison of egt sequence data identified PhopGV-SA as a type II egt gene. A phylogenetic analysis of egt amino acid sequences grouped all type II genes, including PhopGV-SA, into a separate clade from types I, III, IV and V. These findings suggest that type II may represent the prototype structure for this gene with the evolution of types I, III and IV a result of large internal deletion events and subsequent divergence. PhopGV-SA was also shown to be genetically more similar to South American isolates (i.e. PhopGV-CHI or PhopGV-INDO) than it is to other African isolates, suggesting that the South African isolate originated from South America. Restriction endonuclease profiles of PhopGV-SA were similar to those of PhopGV-1346 and PhopGV-JLZ9f for the enzymes BamHI, HindIII, NruI and NdeI. A preliminary full genome sequence for PhopGV-SA was determined and compared to PhopGV-136 with some gene variation observed (i.e. odv-e66 and vp91/p95). The biological activity of PhopGV-SA against P. operculella neonate larvae was evaluated with an estimated LC₅₀ of 1.87×10⁸ OBs.ml⁻¹ being determined. This study therefore reports the characterisation of a novel South African PhopGV isolate which could potentially be developed into a biopesticide for the control of P. operculella.
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Showing items 1 - 20 of 25

Evaluating baculovirus mixtures against false codling moth Thaumatotibia leucotreta Meyrick. (Lepidoptera: Tortricidae)

Investigation into methods of recovering campylobacter spp. from river water samples

The development and op timisation of a Theiler’s murine encephalomyelitis virus antiviral assay

Development and optimisation of a qPCR assay for the enumeration of Cryptophlebia leucotreta granulovirus (CrleGV) used for commercial applications

An investigation into yeast-baculovirus synergism for the improved control of Thaumatotibia leucotreta, an economically important pest of citrus

Baculovirus synergism for improved management of false codling moth Thaumatotibia leucotreta Meyr. (Lepidoptera: Tortricidae)

Selection for improved virulence of Cryptophlebia peltastica nucleopolyhedrovirus (CrpeNPV) to False Codling Moth, Thaumatotibia leucotreta, by serial passage through a heterologous host

Exploring the structural integrity of a picornavirus capsid

A computational analysis to decipher the pathways of stability, uncoating and antigenicity of human enterovirus capsids

Genetic characterisation of a range of geographically distinct Helicoverpa armigera nucleopolyhedrovirus (HearNPV) isolates and evaluation of biological activity against South African populations of the African bollworm, Helicoverpa armigera (Hu bner) (Lepidoptera: Noctuidae)

Recovery and molecular identification of Aichi virus 1, enteric human bocaviruses and enteric human adenoviruses in untreated sewage and mussel samples collected in the Eastern Cape Province of South Africa

Baculovirus synergism: investigating mixed alphabaculovirus and betabaculovirus infections in the false codling moth, thaumatotibia leucotreta, for improved pest control

Isolation, identification and genetic characterisation of a microsporidium isolated from the carob moth, Ectomyelois ceratoniae (Lepidoptera: Pyralidae)

Yeast-baculovirus synergism: investigating mixed infections for improved management of the false codling moth, Thaumatotibia leucotreta

The development of techniques for the identification of novel viruses associated with acute infantile gastroenteritis in South Africa

The isolation and genetic characterisation of a novel alphabaculovirus for the microbial control of Cryptophlebia peltastica and closely related tortricid pests

Genetic and biological characterisation of a novel South African Cydia pomonella granulovirus (CpGV-SA) isolate

Prediction of interacting motifs within the protein subunits of Picornavirus capsids

Production of Cydia pomonella granulovirus (CpGV) in a heteralogous host, Thaumatotibia Leucotreta (Meyrick) (False codling moth)

The isolation, genetic characterisation and biological activity of a South African Phthorimaea operculella granulovirus (PhopGV-SA) for the control of the Potato Tuber Moth, Phthorimaea operculella (Zeller)